1. Field of the Invention
The present invention relates generally to the use of terminal-phosphate-labeled nucleotides with three or more phosphates as substrates for various enzymes including nucleic acid polymerases in the presence of Mn++ as cofactor for enhanced activity. The labels employed are chemiluminescent, fluorescent, electrochemical and chromogenic moieties as well as mass tags and include those that are directly detectable, as well as those that are detectable after enzyme activation or feed into other processes to generate a different signal. These terminal phosphate labeled nucleotides could be used in homogenous assays including identification of specific genotypes or genetic sequences. Further provided are new terminal phosphate labeled nucleoside polyphosphates with linkers that connect the label to the terminal phosphate of the nucleotides to enhance their substrate properties, i.e. to increase their rate of utilization by different enzymes.
2. Description of Related Art
A number of enzymes utilize or act on nucleoside triphosphates and their analogs. These enzymes include ligases, nucleic acid polymerases, phosphodiesterases, phosphorylases, kinases and phosphatases. All these enzymes require certain metal ions as cofactors and although many of these enzymes can work in the presence of different metal ions, one metal is generally more favored than the rest.
Nucleic acid polymerases are enzymes which catalyze the polymerization of nucleoside 5′-triphosphates to form DNA or RNA. They include DNA dependent DNA polymerases, DNA dependent RNA polymerases and RNA dependent DNA polymerases. These enzymes require the presence of a divalent metal ion as cofactor, typically Mg2+, although it is known that Mn2+ and even Ca2+ can sometimes substitute for the Mg2+. With normal nucleotides, the rate of reaction is greater when Mg2+ is used than it is with other metals.
There have been numerous nucleotide analogs made which can also serve as substrates for nucleic acid polymerases, including compounds that have, for example, fluorescent labels. These labels have been attached to the base portion of the nucleotide, and in rare cases to the sugar portion of the nucleotide. These have worked as substrates for polymerases more or less under the same reaction conditions with the same divalent metal ion cofactors as the normal, unsubstituted nucleotides.
There is also interest in the use of nucleotides labeled on the gamma phosphate position. A number of γ-phosphate labeled nucleoside triphosphates are commercially available from Molecular Probes (U.S. Pat. No. 6,323,186). In these nucleotides the dye (bodipy) is attached to the y-phosphate through a phosphorothioate linkage. U.S. Pat. No. 6,399,335 entitled ‘γ-phosphoester nucleoside triphosphates’ provides methods and compositions for polymerizing particular nucleotides with a polymerase using y-phosphoester linked nucleoside triphosphates. A number of other patent applications from LI-COR Inc. (U.S. Pat. No. 6,232,075; US 2003/0194740: WO 01/94609; and US 2003/0186255) describes the synthesis and methods of use of different base and γ-phosphate labeled nucleoside-5′-triphosphates. Using these nucleotides with DNA polymerase can lead to identification of specific nucleotides in a DNA or RNA sequence by identification of the labeled pyrophosphate released upon incorporation of the nucleotide base into RNA or DNA.
Methods are known for detecting specific nucleic acids or analytes in a sample with high specificity and sensitivity. Such methods generally require first amplifying nucleic acid sequence based on the presence of a specific target sequence or analyte. Following amplification, the amplified sequences are detected and quantified. Conventional detection systems for nucleic acids include detection of fluorescent labels, fluorescent enzyme-linked detection systems, antibody-mediated label detection, and detection of radioactive labels.
One disadvantage of these methods is that the labeled product not only requires some type of separation from the labeled starting materials but also since the label is attached to the product, it is different than the natural product to be identified. It would, therefore, be of benefit to use methods and substrates that form unmodified product and at the same time generate a signal characteristic of the reaction taking place. It is of further benefit if the signal generated doesn't require separation from the starting materials but even if a separation is required benefits of generating unmodified product in many cases are overwhelming.
Gamma phosphate labeled nucleotides do provide such an opportunity. For example, incorporation of gamma phosphate labeled nucleotides into DNA or RNA by nucleic acid polymerases results in the production of unmodified DNA or RNA and at the same time the labeled pyrophosphate generated is used to detect, characterize and/or quantify the target. These nucleotides, however, are very poor substrates for various nucleic acid polymerases and it would, therefore be of benefit to improve the rate of incorporation of these terminal-phosphate labeled nucleotides. As demonstrated earlier (WO 03/020891; WO 03/020984; WO 03/020734) some rate enhancement can be achieved by increasing the length of polyphosphate chain between the label and the nucleotide. This rate enhancement although useful for some applications, for practical reasons is still insufficient for many applications where several hundred nucleotides have to be added in a short time, e.g. PCR.
It would, therefore, be of benefit to further enhance the rate of utilization of terminal phosphate labeled nucleotides. This and other concerns are addressed below.